Heat-resisting xylanase and gene coding the xylanase

A xylanase, gene technology, applied in the field of xylanase, can solve problems such as industrial application limitations

Inactive Publication Date: 2005-09-14
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Xylanase can be produced by a variety of microorganisms, but because the xylanase produced by general microorganisms is medium-low temperature or slightly acidic, its industrial application is greatly limited

Method used

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  • Heat-resisting xylanase and gene coding the xylanase
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  • Heat-resisting xylanase and gene coding the xylanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Extraction of Thermotoga maritima Genomic DNA

[0026] 20 g of fresh wet bacteria of Thermotoga maritima (Thermotoga maritima MSB8, German Culture Collection, DSM3109) were used to suspend in 10 mL of 50 mM Tris buffer (pH 8.0), and a small amount of lysozyme and 8 mL of 0.25 mM EDTA (pH 8. 0), mix well and place at 37°C for 20min, then add 2mL of 10% SDS, place at 55°C for 5min, extract once with equal volumes of phenol and chloroform respectively, take the last supernatant, add 2 times the volume of ethanol, DNA recovery. Then wash with 70% and absolute ethanol respectively, dissolve the precipitate in 0.5mL TE buffer (pH8.0, 10mMTris, 1mM EDTA), add 10mg / mL RNase 3μL, keep it at 37°C for 1h, and extract with equal volumes of phenol and chloroform respectively. Once, 2 times the volume of ethanol was added to the supernatant, and the DNA was recovered, washed with 70% ethanol and absolute ethanol, vacuum freeze-dried, and dissolved in ultrapure water.

Embodiment 2

[0027] Embodiment 2 Amplification of xylanase gene xynB

[0028] A pair of primers were designed, and the Nco I-Hind III restriction sites capable of being inserted into the pET28a(+) plasmid (Novagen) were introduced into the primers. The sequences of the primers TM-XynB-Nco I-FWD and TM-XynB-Hind-His-REV used are as follows:

[0029] TM-XynB-Nco I-FWD: 5′-CCATGGAAA TATTACCTTCTGTGTGAT

[0030] TM-XynB-Hind-His-REV: 5′-AAGCTTTCTTTCTTCTATCTTTTTCTCCA

[0031]When designing the TM-XynB-Nco I-FWD forward primer, it is considered that the C-terminus in the expression vector has a 6×His tag, so that the second amino acid from the Nco I restriction site is changed from K to E after translation. This mutation according to the primer design ensures that the target gene is cloned in the open reading frame, with ATG start codon and 6×His tag sequence at the same time, without open reading frame drift.

[0032] Using the genomic DNA of Thermotoga maritima as a template, polymerase was ...

Embodiment 3

[0034] Cloning and DNA sequence determination of embodiment 3 PCR products

[0035] After the PCR amplification reaction, the present invention uses topological TA cloning to clone the xynB gene fragment into the pCR(R)-2.1Topo plasmid vector, and screens colonies with recombinants by colony PCR. Then, use the standard primer sequence on the pCR(R)-2.1Topo plasmid vector as a primer to measure the DNA sequence of the xynB gene, and purify the plasmid containing the xynB gene sequence for further subcloning and expression.

[0036] The present invention adopts terminal termination method (capillary electrophoresis fluorescence method) for sequencing, and the reagent used for sequencing is the Dye Terminor Cycle sequencing kit (Perkin-Elmer, USA). The composition of the sequencing reaction mixture is: Terminator Ready Reaction Mix, 4μl; Plasmid Solution, 3μl; Forward or Reverse primer, 1.5μl; H 2 O, 1.5 μl; the total volume of the reaction mixture was 10 μl. The sequencing cyc...

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Abstract

The present invention discloses xylanase B gene obtained with Thermotoga maritime MSB8 genome DNA and through PCR amplification and the construction of expression vector to express the gene encoded recobinant xylanase in colibacillus. The xylanase has optimal reaction temperature of 90 deg.c, high heat stability in neutral and slight alkali environment, and activity as high as over 1100 times that of cellulase. The xylanase exhibits excellent application foreground as bleaching assistant in pulp producing and papermaking industry. The main product of hydrolyzing xylan is xylobiose, and the present invention is suitable for decomposing xylan in hemicellulose to produce functional oligoxylan.

Description

field of invention [0001] The invention relates to a xylanase, a gene encoding the enzyme and an expression vector containing the gene. Background of the invention [0002] Hemicellulose is the second most abundant available natural resource after cellulose, and its main component is xylan. β-1,4-endo-xylanase (EC.3.2.1.8) can act on the main chain of xylan in an endo-cutting manner to produce xylooligosaccharides of different lengths and a small amount of xylose, and is one of the xylan degrading enzymes most critical enzymes. Xylanase can be widely used in biotransformation, food, feed, medicine, energy, papermaking, textile and other industries. Xylanase can be produced by a variety of microorganisms, but because the xylanase produced by general microorganisms is medium-low temperature or slightly acidic, its industrial application is greatly limited. [0003] Heat-resistant or high-temperature-resistant xylanases have many advantages in industrial applications, so the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63
Inventor 江正强李里特李颖林清
Owner CHINA AGRI UNIV
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